A common thread in all forms of diabetes is ? cell failure. Data have identified ? cell signaling pathways activated during diabetes development that exacerbate ? cell failure and death. Defining these pathways and their contribution to diabetes will ultimately pave the way for novel ? cell targeted biomarkers and therapies. Extracellular vesicles (EVs) are membrane bound nanoparticles that can be transferred to other cells as a means of cell:cell communication. Emerging data suggest that ? cell-derived exosomes, an EV subtype released by exocytosis of multivesicular bodies, and their cargo may act as paracrine effectors in islet health. However, the mechanisms linking ? cell stress to changes in exosome cargo and whether activation of these pathways can impact diabetes progression remain unexplored. The central hypothesis of this application is that DNA damage induced by islet inflammatory stress activates signaling pathways regulating ? cell exosome miRNA cargo, initiating a cascade of ? cell death and dysfunction through exosomal miRNA transfer to surrounding ? cells. Aim 1 will elucidate the mechanistic etiology of cytokine-induced alterations in ? cell exosome microRNA cargo. We hypothesize that cytokine-induced ? cell DNA damage and p53 activation engage ceramide-dependent exosomal formation pathways. This process ultimately increases ceramide-enriched exosome subpopulations, and drives critical differences in exosome miRNA cargo. To test this, experiments will utilize chemical and genetic manipulation of ? cell DNA damage, p53 activation, and ceramide generation. Aim 2 will utilize in vitro and in vivo models to determine the functional relevance of ? cell exosome microRNA cargo transfer to other ? cells. We hypothesize that cytokine-induced alterations in ? cell exosome miRNA cargo exacerbate ? cell death, dedifferentiation, and dysfunction by exosomal transfer of miRNAs to surrounding ? cells. Aim 3 will utilize a bead based pulldown to enrich human circulating EV pools for islet cell-derived EVs. We hypothesize that proteins specific to ? cells will also be present in ? cell EVs, therefore enabling selective isolation of ? cell-derived EVs. We will test whether human subjects with or at-risk for diabetes exhibit altered circulating islet cell-derived EV miRNAs. Functional experiments will define differential effects of incubation of circulating islet-derived EVs on recipient human ? cells. This work will lead to a paradigm shift in the research community's understanding of ? cell:? cell communication, and determine the clinical biomarker potential of islet-derived EV cargo in diabetes.